Apoptosis has been implicated in controlling the amount and distribution of certain differentiated cell types, such as cells of the hematopoietic lineage, as well as other somatic and germ cells. Apoptosis was first described as a morphologic pattern of cell death characterized by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation (Kerr et al., 1972, Br. J. Cancer 26:239). Cells undergoing apoptosis display a characteristic pattern of internucleosomal DNA cleavage (Wyllie, 1980, Int. Rev. Cytol. 69:251; Abrams et al., 1993, Development 117:29).
The first gene to be identified which encoded a protein involved in modulating apoptosis, bcl-2, was cloned from the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas (Tsujimoto et al., 1984, Science 226:1097) and shown to inhibit cellular susceptibility to apoptosis (Cory, 1994, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 345: 289). Several genes with homology to bcl-2 have subsequently been characterized, including the following: a1, which encodes an 80-amino acid protein that is rapidly induced in macrophages in response to GM-CSF or LPS (Lin et al., 1993, J. Immunol. 151: 1979-1988); mcl-1, an early response gene in myeloid cell lines which undergo macrophage differentiation (Kozopas et al., 1993, Proc. Natl. Acad. Sci. USA 90: 3516-3520); and bak, a bcl-2 homologue that may enhance apoptosis (Chittenden et al., 1995, Nature 374:733; Kiefer et al., 1995, Nature 374:736). Other proteins which interact with and/or are structurally related to the bcl-2 gene product have also been identified, such as for example, Bcl-xL and Bcl-xS (Boise et al., 1993, Cell 74:597); Ced-9 (Vaux et al., 1992, Science 258:1955) and two DNA virus proteins, LMW5-HL and BHRF-1 of the Epstein Barr virus.
The bcl-x gene product, closely related to the Bcl-2 protein family, also protects cells from apoptosis. Analysis of mice deficient in Bcl-x suggests that it supports the viability of immature cells during development of the nervous and hematopoietic systems (Motoyama et al., 1995, Science 267: 1506-1510; Ma et al., 1995. Proc. Natl. Acad. Sci. USA 92: 4763-4767). Alternative splicing of human bcl-x may result in at least two distinct bcl-x mRNA species, bcl-xL and bcl-xS. The predominant protein product (233 amino acids) of the larger bcl-x mRNA, Bcl-xL, inhibits cell death upon growth factor withdrawal (Boise et al., 1993. Cell 74, 597-608) and its transgenic expression alters thymocyte maturation leading to increased numbers of mature thymocytes (Chao et al., 1995, J. Exp. Med. 182: 821-828; Grillot et al., 1995, J. Exp. Med. 182: 1973-1983). Bcl-xS, on the other hand, inhibits the ability of Bcl-2 to inhibit cell death and renders cells more susceptible to apoptotic cell death. Additional murine Bcl-x isoforms, termed Bcl-xxcex2 and Bcl-xxcex94TM, have been identified. The xcex2 isoform may inhibit apoptosis in neurons (Gonzalez-Garcia et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92: 4304-4308) and the xcex94TM isoform may inhibit apoptosis in B-cells (Fang et al., supra).
The BCL-2 family of proteins is thus comprised of pro-apoptotic as well as anti-apoptotic members (Farrow and Brown, 1996, Curr. Opin. Genet. Dev. 6:45). Bcl-2-related proteins share homology clustered within four conserved regions termed Bcl-2 homology 1 through 4 (BH1-4) domains. An amphipathic alpha helix, BH3, is of particular importance for the proapoptotic family members (Korsmeyer, 1999, Cancer Res. 1: 1693s-1700s; Chittenden et al., 1995, EMBO J. 14: 5589). Proapoptotic molecules bear sequence homology to the Bcl-2 family only at BH3. A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3 -containing death agonists (Minn et al., EMBO J. 1999, 18 (3): 632-43).
In addition to playing a role in normal development, apoptosis has been implicated in pathologic conditions, such as Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (see e.g., Passer et al., J. Biol. Chem. 1999, 274: 24007). Moreover, the ability to modulate apoptosis in cells would be valuable in controlling undesirable cell proliferation, e.g., the proliferation of cancer cells. Thus, the identification of agents that can modulate apoptosis may be useful in controlling cell proliferation, differentiation, and/or apoptosis in research and therapeutic applications.
The present invention is based, at least in part, on the discovery that Pablo and polypeptides derived therefrom interact with Bcl-xL and, therefore, are useful as modulating agents in regulating a variety of cellular processes, particularly in neural cell processes.
Accordingly, in one aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated mammalian Bcl-xL binding domain, wherein said isolated mammalian Bcl-xL binding domain has 70% amino acid sequence identity with a Bcl-xL binding domain set forth in SEQ ID NO:2.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated mammalian Bcl-xL binding domain, wherein said nucleotide sequence hybridizes to the complement a nucleotide sequence set forth in SEQ ID NO:1 which encodes a Bcl-xL binding domain in 6xc3x97SSC at 45xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0.1% SDS at 50-65xc2x0 C.
In one embodiment, the isolated Bcl-xL binding domain consists of amino acids 419-559 or amino acids 429-559 of SEQ ID NO:2. In another embodiment, the isolated mammalian Bcl-xL binding domain modulates apoptosis in a neural cell.
In one embodiment, the nucleic acid molecule is in a vector.
In one embodiment, the nucleic acid molecule comprises a naturally-occurring nucleotide sequence.
In one embodiment, the nucleic acid molecule encodes a fusion protein.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a first polypeptide comprising a mammalian Bcl-xL binding domain, wherein said Bcl-xL binding domain has 70% amino acid sequence identity with a Bcl-xL binding domain set forth in SEQ ID NO:2 and a second peptide comprising non-Pablo a amino acid sequence.
In yet another aspect, the invention provides a polypeptide comprising an isolated mammalian Bcl-xL binding domain, wherein said isolated Bcl-xL binding domain consists of an amino acid sequence having at least 70% identity with a Pablo Bcl-xL binding domain shown in SEQ ID NO:2.
In still another embodiment, the invention provides a polypeptide comprising a Bcl-xL binding domain, wherein said Bcl-xL binding domain consists of an amino acid sequence having at least 70% identity with a Pablo Bcl-xL binding domain shown in SEQ ID NO:2, provided said polypeptide is not a full-length Pablo polypeptide.
In yet another aspect, the invention is directed to polypeptide comprising an isolated Bcl-xL binding domain set forth in SEQ ID NO:2.
In one embodiment, the invention is directed to a polypeptide in which a conservative amino acid substitution has been made.
In one aspect, the invention is drawn to a polypeptide consisting of an isolated Bcl-xL binding domain set forth in SEQ ID NO:2.
In one embodiment, the isolated Bcl-xL binding domain consists of amino acids 419-559 or amino acids 429-559.
In one embodiment, the isolated Bcl-xL binding domain modulates apoptosis in a neural cell.
In one aspect, the invention is directed to a fusion protein comprising a first polypeptide consisting of an isolated Bcl-xL binding domain and a second, non-Pablo polypeptide.
In yet another aspect, the invention is directed to an isolated nucleic acid molecule which is antisense to the portion of SEQ ID NO:1 which encodes a Bcl-xL binding domain.
In still another aspect, the invention is directed to a vector comprising a nucleic acid molecule of the invention. In one embodiment, such a vector is contained in a host cell.
In yet another aspect, the invention is directed to a neural cell line stably expressing a heterologous Pablo polypeptide or an isolated Bcl-xL binding domain set forth in SEQ ID NO:2.
In still another aspect, the invention is drawn to a nonhuman transgenic animal which contains cells carrying a nucleic acid molecule encoding an isolated mammalian Bcl-xL binding domain.
In yet another aspect, the invention is directed to a method of modulating apoptosis in a cell comprising modulating the activity of a Pablo polypeptide or Bcl-xL binding domain thereof.
In one embodiment, the step of modulating the activity of a Pablo polypeptide comprises modulating the interaction of a Bcl-xL binding domain with a Bcl-xL molecule.
The invention is further directed to a method of modulating apoptosis in a cell comprising modulating the expression of a Pablo polypeptide or Bcl-xL binding domain thereof.
In still another aspect, the invention is directed to a method for modulating Pablo activity in a cell comprising contacting the cell with an agent that modulates Pablo activity such that Pablo activity in the cell is modulated.
In yet another aspect, the invention is drawn to a method for modulating Pablo expression in a cell comprising contacting the cell with an agent that modulates Pablo expression such that Pablo expression in the cell is modulated.
In one embodiment, apoptosis is modulated in the cell.
In another embodiment, the cell is a neural cell.
In yet another aspect, the invention is directed to a method for treating a nervous system disorder in a subject comprising modulating the expression or activity of Pablo in a cell of the subject to thereby treat a nervous system disorder in the subject.
In another aspect, the invention is directed to a method for detecting the presence of Pablo in a cell comprising contacting the cell with an agent that detects expression or activity of Pablo thereby detecting Pablo in the cell.
In yet another aspect, the invention is drawn to a method for identifying a compound that modulates the pro-apoptotic activity of a Bcl-xL binding domain, comprising:
contacting a cell expressing a Bcl-xL binding domain with a test compound and;
determining the ability of the test compound to modulate the activity of a Bcl-xL binding domain to thereby identify a compound that modulates the pro-apoptotic activity of a Bcl-xL binding domain.
In still another aspect, the invention is directed to a method for identifying a compound that modulates the pro-apoptotic activity of a Bcl-xL binding domain, comprising:
contacting a cell-free mixture comprising a Bcl-xL binding domain with a test compound and;
determining the ability of the test compound to modulate the activity of a Bcl-xL binding domain to thereby identify a compound that modulates the pro-apoptotic activity of a Bcl-xL binding domain.
In one embodiment, the cell is a neural cell.
In another embodiment, the cell-free system comprises isolated mitochondria.
In one embodiment, the activity of a Bcl-xL binding domain is assayed by measuring the ability of the Bcl-xL binding domain to bind to Bcl-xL